Metabolic engineering of Escherichia coli to efficiently produce monophosphoryl lipid A.

Monophosphoryl lipid A (MPL), mainly isolated from Salmonella minnesota R595, has been used as adjuvant in several vaccines. In this study, an Escherichia coli strain that can efficiently produce the MPL has been constructed. The gene clusters related to the biosynthesis of O-antigen, core oligosaccharide, enterobacterial common antigen, and colanic acid were sequentially removed to save the carbon source and to increase the activity of PagP in E. coli MG1655. Then, the genes pldA, mlaA, and mlaC related to the phospholipid transport system were further deleted, resulting in the strain MW012. Finally, the genes lpxE from Francisella novicida and pagP and pagL from Salmonella were overexpressed in MW012 to modify the structure of lipid A, resulting in the strain MW012/pWEPL. Lipid A species were isolated from MW012/pWEPL and analyzed by thin-layer chromatography and liquid chromatography-mass spectrometry. The results showed that mainly two MPL species were produced in E. coli MW012/pWEPL, one is hexa-acylated, and the other is penta-acylated. More importantly, the proportion of the hexa-acylated MPL, which is the most effective component of lipid A vaccine adjuvant, reached 75%. E. coli MW012/pWEPL constructed in this study provided a good alternative for the production of lipid A vaccine adjuvant MPL.

Engineering Escherichia coli MG1655 to Efficiently Produce 3-Deacyl-4'-monophosphoryl Lipid A.

Monophosphoryl lipid A, derived from Salmonella minnesota R595, has been used in various adjuvant formulations. Escherichia coli can produce lipid A, but its structure is different. In this study, E. coli MG1655 has been engineered to efficiently produce the monophosphoryl lipid A. First, 126 genes relevant to the biosynthesis of the fimbriae, flagella, and ECA were deleted in MG1655, resulting in WQM027. Second, the genes pldA, mlaA, and mlaC related to the phospholipid transport system, the gene ptsG related to the carbohydrate phosphotransferase system, and the gene eptA encoding phosphoethanolamine transferase for lipid A modification were further deleted from WQM027, resulting in MW020. Third, lpxE from Francisella novicida and pagP and pagL from Salmonella were overexpressed in pFT24, resulting in pTEPL. pTEPL was transformed into MW020, resulting in MW020/pTEPL. Finally, fabI encoding an enoyl-ACP reductase was deleted from the genome of MW020/pTEPL, resulting in MW021/pTEPL. MW021/pTEPL could produce 85.31 mg/L of lipid A species after 26 h of fed-batch fermentation. Mainly two monophosphoryl lipid A species were produced in MW021/pTEPL, one is 3-deacyl-2-acyloxyacyl-4'-monophosphoryl lipid A and the other is 3-deacyl-4'-monophosphoryl lipid A. E. coli MW021/pTEPL constructed in this study could be an ideal host for the industrial production of monophosphoryl lipid A.